|
| Status |
Public on Jul 31, 2014 |
| Title |
Daoy_1.5h_SHH+EGF_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
1.5 h, SHH+EGF, rep 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Daoy cells (ATCC: HTB-186)
treatment: SHH+EGF
timepoint: 1.5 h
|
| Treatment protocol |
Hyperconfluent Daoy cells were pre-starved for 24 h in serum-reduced DMEM medium containing 0.5% (v/v) FBS before adding Sonic Hedgehog conditioned medium (Shh-N) or EGF (5 ng/ml) for stimulation. Total RNA was obtained at 14 different time points 24 h after EGF stimulation.
|
| Growth protocol |
Daoy cells (ATCC: HTB-186) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco/Invitrogen) with 10% (v/v) fetal bovine serum (FBS, Gibco/Invitrogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini kit (Qiagen, 74104) according to the manufacturer's protocol. Quantity and purity of RNA were determined by measuring the optical density at 260 and 280 nm with an UV/Vis Spectrophotometer (Thermo Scientific, Nanodrop 1000) and BioAnalyzer 2100 (Agilent Technologies Deutschland GmbH).
|
| Label |
biotin
|
| Label protocol |
500 ng of total RNA in 11 µl RNase free water served as starting material for the generation of biotin-labeled cRNA with the Illumina® TotalPrepTM RNA amplification kit following supplier instructions. cRNA was cleaned up with cRNA filter cartridges before use for subsequent hybridization on Illumina® Sentrix BeadChips.
|
| |
|
| Hybridization protocol |
To perform whole genome expression analysis HumanHT-12 v4 chips were incubated with biotin labeled cRNA for 18h at 58°C in a hybridization oven under humidity controlled conditions. After hybridization the Illumina® Sentrix BeadChips were washed using buffers provided by the kit. 2.5 µl (1mg/ml) of Streptavidin-Cy3 (per Chip) diluted in 2.5 ml Blocking buffer were incubated on a Chip for 10 min under gentle shaking to allow binding of cRNA to gene specific probes. After washing, Illumina® Sentrix BeadChips were dried and and scanned.
|
| Scan protocol |
Standard Illumina scanning protocol
|
| Description |
replicate 3
|
| Data processing |
The data were normalised using quantile normalisation with lumi package in R.
|
| |
|
| Submission date |
Apr 15, 2013 |
| Last update date |
Jul 31, 2014 |
| Contact name |
Christoph Wierling |
| E-mail(s) |
wierling@molgen.mpg.de
|
| Organization name |
Max Planck Institute for Molecular Genetics
|
| Department |
Department Vertebrate Genomics
|
| Lab |
Systems Biology
|
| Street address |
Ihnestr. 73
|
| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10558 |
| Series (1) |
| GSE46045 |
Synergism between Hedgehog-GLI and EGFR signaling in Hedgehog-responsive human medulloblastoma (Daoy) cells |
|
